Methods of treating or preventing zika virus infection

ABSTRACT

This document relates to methods and materials for treating a mammal having a Zika virus (ZIKV) infection. For example, a composition including one or more non-nucleoside RNA polymerase inhibitors can be administered to a mammal having, or at risk of developing, a ZIKV infection to treat the mammal

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Ser. No.62/579,495, filed on Oct. 31, 2017. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials for treating a mammalhaving a Zika virus (ZIKV) infection. For example, one or morenon-nucleoside RNA polymerase inhibitors can be administered to a mammalhaving, or at risk of developing, a ZIKV infection to treat the mammal.

2. Background Information

ZIKV, a mosquito-borne pathogen, was originally isolated in Uganda in1947 (Dick et al., 1952 Trans. Roy. Soc. Trop. Med. Hyg. 46:509-520) andonly sporadic cases of virus outbreaks in humans were reported in Africaand Asia in the next six decades (Mecharles et al., 2016 Lancet387:1481; Munoz et al., 2016 Semin. Reprod. Med. 34:273-279). However,in the past ten years, it has rapidly emerged and spread to the regionsof Asia, Europe, and the Americas (Aliota et al., 2017 Antivir. Res.144:223-246; Chan et al., 2016 J. Infect. 72:507-524; Deseda, 2017 Curr.Opin. Pediatr. 29:97-101; Weaver et al., 2016 Antivir. Res. 130:69-80).Although the majority of infections in humans are asymptomatic, recentZIKV infections have been linked to a variety of congenital disordersincluding microcephaly and fetal growth restriction (Carteaux et al.,2016 N. Engl. J. Med. 374:1595-1596; Cauchemez et al., 2016 Lancet387:2125-2132; Chan et al., 2016 J. Infect. 72:507-524; Coyne andLazear, 2016 Nat. Rev. Microbiol. 14:707-715; Cugola et al., 2016 Nature534:267-271; Lazear and Diamond, 2016 J. Virol. 90:4864-4875; Miner andDiamond, 2017 Cell Host Microbe 21:134-142; and Mlakar et al., 2016 N.Engl. J. Med. 374:951-958) as well as Guillain-Barre syndrome in adults(Avelino-Silva and Martin, 2016 Lancet 387:2599; Nascimento et al., 2017Neurology 88:2330-2332; and Parra et al., 2016 N. Engl. J. Med.375:1513-1523). These severe consequences and the large-scale spreadingof the virus have imposed a significant threat to human health worldwide(Fauci and Morens, 2016 N. Engl. J. Med. 374:601-604; Gulland, 2016 BMJ352:i657; Roos, 2016 J. Neurol. 73:1395-1396). So far, no vaccine ordrug for preventing or treating this viral disease is available (Shan etal., 2016 Adv. Infect. Dis. 2:170-172). Therefore, it is urgent todevelop countermeasures against this viral epidemic (Rather et al., 2017Front. Microbiol. 8:305; Salam et al., 2017 Ann. Intern. Med.166:725-732).

SUMMARY

ZIKV has become a major human health concern globally due to itsassociation with congenital abnormalities and neurological diseases.

This document provides methods and materials for treating a mammalhaving, or at risk of developing, ZIKV in its bloodstream (e.g., ZIKVviremia). In some cases, ZIKV viremia can lead to a ZIKV infection. Forexample, one or more non-nucleoside RNA polymerase inhibitors (e.g.,3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB)) can be administered to a mammal having, or at risk of developing,ZIKV viremia to treat the mammal. In some cases, one or morenon-nucleoside RNA polymerase inhibitors can inhibit ZIKV replication(e.g., within in a cell in a mammal). In some cases, one or morenon-nucleoside RNA polymerase inhibitors can reduce ZIKV viremia in amammal.

As demonstrated herein, TPB inhibited ZIKV replication at sub-micromolarconcentrations (e.g., the half-maximal inhibitory concentration (IC₅₀)and the cytotoxicity concentration (CC₅₀) of TPB in Vero cells were 94nM and 19.4 μM, respectively, yielding a high selective index 50 (SI₅₀)of 206). Without being bound by theory, molecular docking analysissuggested that TPB binds to the catalytic active site of the ZIKVRNA-dependent RNA-polymerase (RdRp) and therefore likely blocks theviral RNA synthesis by an allosteric effect. Also as demonstratedherein, TPB reduced ZIKV viremia significantly in immunocompetent mice.The ability to inhibit ZIKV replication can reduce ZIKV viremiaproviding a unique and unrealized opportunity to treat and/or preventZIKV infections. For example, TPB can be used to treat and/or preventZIKV infections.

In general, one aspect of this document features methods for treatingmammals having a ZIKV infection. The methods can include, or consistessentially of, administering to a composition including anon-nucleoside RNA polymerase inhibitor to a mammal having a ZIKVinfection to treat the mammal. The mammal can be a human. Thenon-nucleoside RNA polymerase inhibitor can bind to a catalytic activesite of an RdRp of a ZIKV to inhibit ZIKV replication. Thenon-nucleoside RNA polymerase inhibitor can be TPB. The non-nucleosideRNA polymerase inhibitor can have an IC₅₀ of from about 10 nM to about200 nM. The non-nucleoside RNA polymerase inhibitor can have a CC₅₀ offrom about 15 μM to about 25 μM. The non-nucleoside RNA polymeraseinhibitor can have a SI₅₀ of about 206. The administering step can beperformed prior to the mammal being infected with the ZIKV or after themammal being infected with the ZIKV. The administering step can beperformed prior to the mammal being infected with the ZIKV and after themammal being infected with the ZIKV. The non-nucleoside RNA polymeraseinhibitor can be administered intraperitoneally, intravenously,intramuscularly, or subcutaneously.

In another aspect, this document features methods for method ofpreventing microcephaly in a fetus. The methods can include, or consistessentially of, administering a composition including a non-nucleosideRNA polymerase inhibitor to a mammal pregnant with a fetus, where thepregnant mammal has a ZIKV infection. The mammal can be a human. Thenon-nucleoside RNA polymerase inhibitor can bind to a catalytic activesite of an RdRp of a ZIKV to inhibit ZIKV replication. Thenon-nucleoside RNA polymerase inhibitor can be TPB. The non-nucleosideRNA polymerase inhibitor can have an IC₅₀ of from about 10 nM to about200 nM. The non-nucleoside RNA polymerase inhibitor can have a CC₅₀ offrom about 15 μM to about 25 μM. The non-nucleoside RNA polymeraseinhibitor can have a SI₅₀ of about 206. The non-nucleoside RNApolymerase inhibitor can be administered intraperitoneally,intravenously, intramuscularly, or subcutaneously.

In another aspect, this document features methods for treating adultmammals having Guillain-Barre syndrome. The methods can include, orconsist essentially of, administering a composition including anon-nucleoside RNA polymerase inhibitor to a mammal having a ZIKVinfection and having Guillain-Barre syndrome to treat the mammal. Themammal can be a human. The non-nucleoside RNA polymerase inhibitor canbind to a catalytic active site of an RdRp of a ZIKV to inhibit ZIKVreplication. The non-nucleoside RNA polymerase inhibitor can be TPB. Thenon-nucleoside RNA polymerase inhibitor can have an IC₅₀ of from about10 nM to about 200 nM. The non-nucleoside RNA polymerase inhibitor canhave a CC₅₀ of from about 15 μM to about 25 μM. The non-nucleoside RNApolymerase inhibitor can have a SI₅₀ of about 206. The non-nucleosideRNA polymerase inhibitor can be administered intraperitoneally,intravenously, intramuscularly, or subcutaneously.

In another aspect, this document features compositions for reducing ZIKVviremia within a mammal. The compositions include a non-nucleoside RNApolymerase inhibitor. The non-nucleoside RNA polymerase inhibitor can beTPB. The non-nucleoside RNA polymerase inhibitor can bind to a catalyticactive site of an an RdRp of a ZIKV. The non-nucleoside RNA polymeraseinhibitor can have an IC₅₀ of from about 10 nM to about 200 nM. Thenon-nucleoside RNA polymerase inhibitor can have a CC₅₀ of from about 15μM to about 25 μM. The non-nucleoside RNA polymerase inhibitor can havea SI₅₀ of about 206. The composition also can include a pharmaceuticallyacceptable carrier.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a structure-based screening of inhibitors of ZIKV targetingthe viral RNA-dependent RNA-polymerase (RdRp). (A) Structure of ZIKVRdRp generated by homology modeling. The three subdomains are coloredindividually, Fingers (cyan), Thumb (blue) and Palm (red). The catalyticactive site and the priming loop are labeled. Docking of TPB on theactive site of RdRp and the contacts with the two aspartic residues(D535 and D665) along with the three hydrogen bonds are indicated. (B)The chemical structure of TPB (molecular weight of 541.108). (C) Alarger view of the boxed area in A is shown for clarity. (D) Thespace-filing model of the boxed area in A is shown along with the boundTPB. TPB binding at the palm subdomain is shown.

FIG. 2 shows inhibition of ZIKV replication by the top 10 leadcompounds. (A) Viral genome copies in the supernatants of cells infectedwith PRVABC59 virus in the presence of 1 μM concentration of the tencompounds (c1-c10) or with vehicle (DMSO) alone for 72 hours. The dataare expressed as % of DMSO-treated control. The experiment was done intriplicate and bars represent ±SEM. (B) Infectious virus titers in thesupernatant of cells infected with the virus and incubated with 1 μMconcentration of the compounds or with vehicle (DMSO) alone for 96hours.

FIG. 3 shows validation of the antiviral effect of TPB in the μM range.(A) Cells infected with PRVABC59 virus were incubated in the presence ofvarious concentrations of TPB for 96 hours. Culture supernatants weretitrated for viral genome copies (A) and infectious virus (B). Data arefrom three independent experiments with error bars showing ±SEM.Statistical analysis was performed using unpaired two-tailed Student'st-test to determine significance of difference. ****, p≤0.001. (C)Western blot analysis of E protein expression in virus-infected cells inthe presence of various concentrations of TPB. Relative mobility ofmolecular mass markers are shown on the left.

FIG. 4 shows TPB inhibition of ZIKV is strain and cell-type independent.(A) Inhibition of MR766 virus by TPB. The experiments were conducted asin FIG. 3B and data from three independent experiments are presentedwith error bars representing SEM. PRVABC59 virus growth in HTR-8 (B) andNTERRA (C) cell lines in the presence of TPB. Data from threeindependent experiments are presented with error bars showing ±SEM.Statistical analysis was performed using unpaired two-tailed Student'st-test to determine significance of difference. ****, p≤0.001.

FIG. 5 shows IC₅₀ and CC₅₀ of TBP. (A) Vero cells in triplicate wereinfected with PRVABC59 virus and incubated with various concentrationsof TPB as shown. Infectious virus titers in the supernatants of thecells at 96 hours post-infection were determined by plaque assay andvirus yield was expressed as % of TPB-untreated control. Non-linearregression analysis was employed to determine the IC₅₀. (B) Vero cellsin triplicate were treated with TPB at various concentrations for fourdays and cell viability was measured based on ATP assay. Theluminescence signals were measured at 420 nm using a MicroplateLuminometer. Non-linear regression analysis of the data was employed todetermine the CC₅₀.

FIG. 6 shows a comparison of TPB inhibitory activity with mycophenolicacid (MPA) and Ivermectin (IVM). Vero cells were infected with PRVABC59virus and incubated with TPB (1 μM), MPA (1 μM) and IVM (10 μM). Culturesupernatants were collected at 96 hours post-infection and assayed forinfectious virus. Data presented are from three independent experimentswith error bars showing ±SEM. Statistical analysis was performed usingunpaired two-tailed Student's t-test to determine significance ofdifference. ***p≤0.01; ****p≤0.001.

FIG. 7 shows inhibitory efficacy of TPB in mice. (A) Pharmacokinetics(PK) analysis of TPB in mice using two different doses as shown. (B)Genome copies at various days post-infection in the plasma of individualmice treated without (continuous lines) or with (discontinuous lines)TPB. (C) Data from the mice groups in panel B. Error bars show ±SEM.Statistical analysis was performed using unpaired two-tailed Student'st-test to determine significance of difference. ****, p≤0.001. (D) TPBmean concentrations in mice plasma at various days post-infection. Errorbars show ±SEM.

FIG. 8 shows model of superimposed structures of ZIKV RdRp. Cyan, RdRpstructure derived from homology modeling; yellow, crystal structure ofRdRp (PDB: 5WZ3). Various domains are identified.

FIG. 9 shows models of an alignment of compounds within the target siteof the ZIKV RdRp (A) and an enlarged view of the positioning of thecompounds (B) in the target site of the RdRp.

FIG. 10 contains exemplary non-nucleoside RNA polymerase inhibitors.

DETAILED DESCRIPTION

This document provides methods and materials for treating a mammalhaving, or at risk of developing ZIKV viremia (e.g., a ZIKV infection).In some cases, this document provides compositions (e.g., pharmaceuticalcompositions such as vaccines) including one or more non-nucleoside RNApolymerase inhibitors (e.g., TPB). In some cases, this document providesmethods for using one or more non-nucleoside RNA polymerase inhibitorsprovided herein to treat a mammal having, or at risk of having, a ZIKVinfection. For example, one or more non-nucleoside RNA polymeraseinhibitors can be administered to a mammal (e.g., a human) having, or atrisk of developing, a ZIKV infection to treat the mammal. In some cases,one or more non-nucleoside RNA polymerase inhibitors can inhibit ZIKVreplication (e.g., within in a cell in a mammal). In some cases, one ormore non-nucleoside RNA polymerase inhibitors can reduce ZIKV viremia ina mammal. One or more non-nucleoside RNA polymerase inhibitors can beadministered to a mammal to protect the mammal from a ZIKV infection(e.g., prior to exposure to a ZIKV) and/or to treat the mammal (e.g.,after exposure to a ZIKV).

Any appropriate mammal (e.g., a mammal having, or at risk of developing,ZIKV viremia) can be treated as described herein. In some cases, amammal can have, or can be at risk of developing, a ZIKV infection. Insome cases, a mammal can carry ZIKV without developing ZIKV infection.Examples of mammals that can be treated as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal) include, without limitation, humans, non-humanprimates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice,rats, horses, cows, carabaos (water buffaloes), goats, ducks, and bats.For example, a human having, or at risk of developing, ZIKV viremia canbe treated by administering one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) to that human. In some cases, a mammal can be apregnant mammal (e.g., pregnant human). When a mammal is a pregnanthuman, the pregnant human can be in any stage of pregnancy (e.g., firsttrimester, second trimester, or third trimester).

When treating a mammal (e.g., a human) having, or at risk of developing,ZIKV viremia (e.g., a ZIKV infection) as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal), the mammal can be any appropriate age. In somecases, a mammal can be an adult. For example, when a mammal is a human,an adult human can be about 18 years of age or older (e.g., about 20years of age, about 30 years of age, about 40 years of age, about 50years of age, about 60 years of age, about 65 years of age, about 70years of age, or about 75 years of age or older). For example, when amammal is a human, an adult human can be from about 18 years of age toabout 80 years of age (e.g., from about 18 years of age to about 60years of age, from about 18 years of age to about 40 years of age, fromabout 25 years of age to about 80 years of age, from about 40 years ofage to about 80 years of age, from about 60 years of age to about 80years of age, from about 20 years of age to about 60 years of age, orfrom about 30 years of age to about 50 years of age). In some cases, amammal can be a juvenile. For example, when a mammal is a human, ajuvenile human can be no more than about 18 years old. For example, ahuman adolescents can be from about 1 year of age to about 18 years ofage (e.g., from about 1 year of age to about 15 years of age, from about1 year of age to about 10 years of age, from about 1 year of age toabout 5 years of age, from about 5 years of age to about 18 years ofage, from about 10 years of age to about 18 years of age, or from about5 years of age to about 15 years of age). In some cases, a mammal can bea newborn. For example, when a mammal is a human, a newborn human fromabout birth to about 1 year of age. In some cases, a mammal can be afetus. For example, when a mammal is a human, a fetus can be in utero(e.g., being carried by a human pregnant with the fetus).

When treating a mammal (e.g., a human) having, or at risk of developing,ZIKV viremia (e.g., a ZIKV infection) as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal), the ZIKV can be any type of ZIKV. A ZIKV can befrom any lineage of ZIKV. A ZIKV can be from any clade of ZIKV. A ZIKVcan be any strain of ZIKV. In some cases, a ZIKV can be a latent ZIKV.In some cases, a ZIKV can be an infectious ZIKV. Examples of ZIKVsinclude, without limitation, East African ZIKV, West African ZIKV, AsianZIKV, and South American ZIKV.

In some cases, methods described herein can include identifying a mammal(e.g., a human) as having a ZIKV infection. Any appropriate method canbe used to identify a mammal having a ZIKV infection. For example, thepresence of a ZIKV in a sample obtained from a mammal can be detected ina sample obtained from a mammal, where the presence of a ZIKV canindicate that the mammal has a ZIKV infection. In some cases, thepresence of a ZIKV genome, or a portion thereof, in a sample obtainedfrom a mammal can be used to identify that mammal (e.g., a human) ashaving a ZIKV infection. In some cases, the presence of one or more ZIKVpolypeptides in a sample obtained from a mammal can be used to identifythat mammal (e.g., a human) as having a ZIKV infection. Any appropriatesample can be assessed to detect the presence of a ZIKV genome, or aportion thereof, and/or the presence of one or more ZIKV polypeptides.For example, biological samples such as fluid samples (e.g., blood(e.g., whole blood, plasma, and serum), urine, breast milk, saliva,amniotic fluid, cerebral spinal fluid, or semen) or tissue samples(e.g., placenta tissue samples) can be obtained from a mammal andassessed for the presence the presence of a ZIKV genome, or a portionthereof, and/or the presence of one or more ZIKV polypeptides. Anyappropriate method can be used to detect the presence the presence of aZIKV genome, or a portion thereof. For example, polymerase chainreaction (PCR) techniques), sequencing techniques, and/or Southernblotting can be used to detect the presence of a ZIKV genome, or aportion thereof, in a sample obtained from a mammal. Any appropriatemethod can be used to detect the presence the presence of one or moreZIKV polypeptides. For example, western blotting techniques,enzyme-linked immunosorbent assays (ELISAs), and/or real-time PCR can beused to detect the presence of one or more ZIKV polypeptides in a sampleobtained from a mammal.

In some cases, methods described herein can include identifying a mammal(e.g., a human) as being at risk of developing ZIKV viremia (e.g., aZIKV infection). For example, a mammal undergoing, or scheduled toundergo, exposure to one or more mammals having ZIKV viremia can be atrisk of developing ZIKV viremia. In some cases, a mammal having physicalcontact (e.g., sexual contact) with one or more mammals having ZIKVviremia can be at risk of developing ZIKV viremia (e.g., a ZIKVinfection). In some cases, a mammal living in or moving to an area whereone or more mammals having ZIKV viremia are present can be at risk ofdeveloping ZIKV viremia (e.g., a ZIKV infection). In some cases, amammal scheduled to travel to an area where one or more mammals havingZIKV viremia are present can be at risk of developing ZIKV viremia(e.g., a ZIKV infection). In some cases, a mammal that has been bitten,or is at risk of being bitten by an animal that carries a ZIKV virus(e.g., a mosquito) can be at risk of developing ZIKV viremia (e.g., aZIKV infection). In some cases, a fetus within a pregnant mammal withZIKV viremia can be at risk of developing ZIKV viremia (e.g., a ZIKVinfection).

A mammal (e.g., a human) identified as having, or as being at risk ofdeveloping, ZIKV viremia (e.g., a ZIKV infection), can be administered,or instructed to self-administer, one or more non-nucleoside RNApolymerase inhibitors. For example, one or more non-nucleoside RNApolymerase inhibitors can be administered to a mammal in need thereof(e.g., a mammal having, or at risk of developing, ZIKV viremia). Anon-nucleoside RNA polymerase inhibitor can be any appropriatenon-nucleoside RNA polymerase inhibitor. A non-nucleoside RNA polymeraseinhibitor can be a chemically synthesized non-nucleoside RNA polymeraseinhibitor. A non-nucleoside RNA polymerase inhibitor can be acommercially obtained non-nucleoside RNA polymerase inhibitor. Examplesof non-nucleoside RNA polymerase inhibitors that can be used asdescribed herein (e.g., to treat a mammal having, or at risk ofdeveloping, ZIKV viremia) include, without limitation, non-nucleosideRNA polymerase inhibitors shown in FIG. 10 (e.g., TPB, C1, C2, C3, C4,C5, C6, C7, C8, C9, and C10). In some cases, a non-nucleoside RNApolymerase inhibitor can be TPB. In cases where a non-nucleoside RNApolymerase inhibitor is TPB, the TPB can be a derivative of TPB. As usedherein, a derivative of a non-nucleoside RNA polymerase can be anystructurally derived compound that maintains the ability to inhibit anon-nucleoside RNA polymerase. For example, a mammal having, or at riskof developing, ZIKV viremia can be administered or can self-administerTPB.

A non-nucleoside RNA polymerase inhibitor (e.g., TPB) can inhibit ZIKVreplication. In some cases, a non-nucleoside RNA polymerase inhibitorcan inhibit transcription of a ZIKV coding sequence (e.g., codingsequence encoding a ZIKV polymerase such as the RdRp polymerase gene).In some cases, a non-nucleoside RNA polymerase inhibitor can inhibitfunction of a ZIKV polypeptide (e.g., a ZIKV polymerase such as the RdRppolymerase). For example, when a non-nucleoside RNA polymerase inhibitoris TPB, the TPB can inhibit function of the RdRp polymerase. In somecases, TPB can target (e.g., bind to) a catalytic active site of theRdRp polymerase to inhibit ZIKV replication.

A non-nucleoside RNA polymerase inhibitor can be a potent inhibitor(e.g., a potent ZIKV inhibitor). For example, when a non-nucleoside RNApolymerase inhibitor is TPB, the TPB can inhibit a ZIKV (e.g., inhibitZIKV replication) at sub-micromolar concentrations. In some cases, theinhibitory concentration 50 (IC₅₀) of TPB can from about 10 nM to about200 nM (e.g., from about 10 nM to about 175 nM, from about 10 nM toabout 150 nM, from about 10 nM to about 125 nM, from about 10 nM toabout 100 nM, from about 10 nM to about 75 nM, from about 10 nM to about60 nM, from about 10 nM to about 50 nM, from about 10 nM to about 40 nM,from about 10 nM to about 30 nM, from about 10 nM to about 20 nM, fromabout 25 nM to about 200 nM, from about 50 nM to about 200 nM, fromabout 70 nM to about 200 nM, from about 90 nM to about 200 nM, fromabout 100 nM to about 200 nM, from about 125 nM to about 200 nM, fromabout 150 nM to about 200 nM, from about 175 nM to about 200 nM, fromabout 25 nM to about 175 nM, from about 50 nM to about 150 nM, fromabout 75 nM to about 125 nM, from about 50 nM to about 100 nM, fromabout 100 nM to about 150 nM, from about 30 nM to about 80 nM, fromabout 50 nM to about 70 nM, or from about 85 nM to about 95 nM). Forexample, the IC₅₀ of TPB can be about 94 nM.

A non-nucleoside RNA polymerase inhibitor can have low toxicity (e.g.,cellular toxicity or cytotoxicity). For example, when a non-nucleosideRNA polymerase inhibitor is TPB, the TPB can have sub-micromolarcytotoxicity concentrations. In some cases, the cellular cytotoxicityconcentration 50 (CC₅₀) of TPB can be from about 15 μM to about 25 μM.For example, the CC₅₀ of TPB can be about 19.4 μM.

A non-nucleoside RNA polymerase inhibitor can have high selectivity(e.g., can be selective for a ZIKV). A selective index 50 (SI₅₀) can bedetermined using the formula CC₅₀/IC₅₀. For example, when anon-nucleoside RNA polymerase inhibitor is TPB, the TPB can have a highSI₅₀. In some cases, the SI₅₀ of TPB can be from about 150 to about 250.For example, the SI₅₀ of TPB can be about 206.

When treating a mammal having, or at risk of developing, ZIKV viremia(e.g., a ZIKV infection), one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be administered to the mammal at anyappropriate time. For example, when a mammal has ZIKV viremia, one ormore non-nucleoside RNA polymerase inhibitors can be administeredbefore, during (e.g., concurrent with), and/or after one or moresymptoms of a ZIKV infection are producing or showing (e.g., after aZIKV infection has developed). In some cases, when a mammal has ZIKVviremia, one or more non-nucleoside RNA polymerase inhibitors can beadministered before one or more symptoms of a ZIKV infection producingor showing no symptoms (e.g., when the mammal is asymptomatic and/orprior to a ZIKV infection developing). For example, when a mammal atrisk of developing ZIKV viremia (e.g., a ZIKV infection) is undergoing,or scheduled to undergo, exposure to one or more mammals having ZIKVviremia (e.g., a ZIKV infection), one or more non-nucleoside RNApolymerase inhibitors can be administered before, during (e.g.,concurrent with), and/or after the exposure.

One or more non-nucleoside RNA polymerase inhibitors (e.g., TPB) can beadministered to a mammal in need thereof (e.g., a mammal having, or atrisk of developing, ZIKV viremia) by any appropriate route.Administration can be local or systemic. Examples of routes ofadministration include, without limitation, intraperitoneal,intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation,transdermal, and parenteral administration. For example, one or morenon-nucleoside RNA polymerase inhibitors can be administeredintraperitoneally to a mammal (e.g., a human).

When treating a mammal having, or at risk of developing, ZIKV viremia(e.g., a ZIKV infection), the treatment can include the administrationof a therapeutically effective amount of one or more non-nucleoside RNAinhibitors. The terms “effective amount” and “therapeutically effectiveamount” refer to that amount of one or more non-nucleoside RNAinhibitors sufficient to result in a therapeutic effect. For example, atherapeutic effect of treating a mammal having, or at risk ofdeveloping, ZIKV viremia can include, without limitation, inhibition ofZIKV replication, reduction or elimination of ZIKV viremia, and/oramelioration (e.g., reduction or elimination) of one or more symptoms ofa ZIKV infection.

In some cases, treating a mammal having, or at risk of developing, ZIKVviremia (e.g., a ZIKV infection) as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal) can be effective to inhibit ZIKV replication. Forexample, administering one or more non-nucleoside RNA polymeraseinhibitors to a mammal can be effective to inhibit ZIKV replicationwithin in one or more cells in that mammal. Any appropriate method canbe used to determine whether or not ZIKV replication has been inhibited.For example, quantitative RT-PCR (RT-qPCR) and/or ELISAs can be used todetermine whether or not ZIKV replication has been inhibited.

In some cases, treating a mammal having, or at risk of developing, ZIKVviremia (e.g., a ZIKV infection) as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal) can be effective to reduce or eliminate ZIKVviremia. For example, administering one or more non-nucleoside RNApolymerase inhibitors to a mammal can be effective to reduce ZIKVviremia within that mammal.

In some cases, administering one or more non-nucleoside RNA polymeraseinhibitors to a mammal having ZIKV viremia can be effective to reduceZIKV viremia by from about 40-fold to about 1000-fold (e.g., from about50-fold to about 1000-fold, from about 80-fold to about 1000-fold, fromabout 100-fold to about 1000-fold, from about 300-fold to about1000-fold, from about 500-fold to about 1000-fold, from about 700-foldto about 1000-fold, from about 800-fold to about 1000-fold, from about900-fold to about 1000-fold, from about 40-fold to about 900-fold, fromabout 40-fold to about 700-fold, from about 40-fold to about 500-fold,from about 40-fold to about 200-fold, from about 40-fold to about100-fold, from about 50-fold to about 900-fold, from about 200-fold toabout 800-fold, from about 500-fold to about 700-fold, from about100-fold to about 400-fold, from about 300-fold to about 600-fold, fromabout 400-fold to about 700-fold, from about 500-fold to about 800-fold,or from about 600-fold to about 900-fold) within that mammal. In somecases, administering one or more non-nucleoside RNA polymeraseinhibitors to a mammal having ZIKV viremia can be effective to reduce aZIKV genome copy number within a mammal. In some cases, administeringone or more non-nucleoside RNA polymerase inhibitors to a mammal havingZIKV viremia can be effective to reduce PFU of ZIKV virus within amammal.

Any appropriate method can be used to determine the presence, absence,or amount of ZIKV in a mammal. For example, RT-qPCR can be used todetermine the presence, absence, or amount of ZIKV in a mammal.

In some cases, treating a mammal having, or at risk of developing, ZIKVviremia (e.g., a ZIKV infection) as described herein (e.g., byadministering one or more non-nucleoside RNA polymerase inhibitors suchas TPB to the mammal) can be effective to reduce the severity of theZIKV infection and/or to reduce or eliminate one or more symptoms of theZIKV infection. In some cases, when a mammal is a pregnant mammal (e.g.,a pregnant human), one or more symptoms can affect the mammal's fetus(e.g., in utero) and/or can affect the mammal's child (e.g., after birthsuch as a newborn child). Examples of symptoms of a ZIKV infection caninclude, without limitation, fever, rash (e.g., maculopapular rash),muscle pain, joint pain, conjunctivitis, vomiting, headache, andcongenital Zika syndrome (e.g., including, but not limited to,microcephaly, decreased brain tissue, damage to the back of the eye suchas scarring and/or pigment changes, joints with limited range of motionsuch as clubfoot, and/or too much muscle tone restricting body movementsoon after birth). In some cases, a symptom of ZIKV infection can be asdescribed elsewhere (see, e.g., www.cdc.gov/zika/symptoms/index.html).For example, treating a pregnant mammal (e.g., a pregnant human),having, or at risk of developing, ZIKV viremia (e.g., a ZIKV infection)as described herein (e.g., by administering one or more non-nucleosideRNA polymerase inhibitors such as TPB to the mammal) can be effective toreduce or eliminate microcephaly in the mammal's fetus and/or themammal's child (e.g., after birth).

In some cases, one or more non-nucleoside RNA polymerase inhibitors(e.g., TPB) can be administered to a mammal having, or at risk ofdeveloping, ZIKV viremia (e.g., a ZIKV infection) in the absence of anycarriers (e.g., additives, fillers, vehicles, and/or diluents).

In some cases, one or more non-nucleoside RNA polymerase inhibitors(e.g., TPB) can be formulated into a composition (e.g., apharmaceutically acceptable composition) for administration to a mammalhaving, or at risk of developing, ZIKV viremia (e.g., a ZIKV infection).For example, one or more non-nucleoside RNA polymerase inhibitors can beformulated together with one or more pharmaceutically acceptablecarriers (e.g., additives, fillers, vehicles, and/or diluents). In somecases, pharmaceutically acceptable carrier can be non-naturallyoccurring. Pharmaceutically acceptable carriers that can be used in apharmaceutical composition described herein include, without limitation,dextrose, methanol, dimethyl sulfoxide (DMSO), ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, and wool fat.

In some cases, a composition including one or more non-nucleoside RNApolymerase inhibitors (e.g., TPB) to be administered to a mammal (e.g.,a human) in need thereof (e.g., a mammal having, or at risk ofdeveloping, ZIKV viremia) can include one or more non-nucleoside RNApolymerase inhibitors as the sole active ingredient. For example, TPBcan be administered to a mammal having, or at risk of developing, ZIKVviremia (e.g., a ZIKV infection) as the sole active ingredient used totreat the mammal.

In some cases, a composition including one or more non-nucleoside RNApolymerase inhibitors (e.g., TPB) to be administered to a mammal (e.g.,a human) in need thereof (e.g., a mammal having, or at risk ofdeveloping, ZIKV viremia) can include one or more non-nucleoside RNApolymerase inhibitors together with one or more additional activeingredients (e.g., active ingredients that can be used to treat a mammalhaving, or at risk of developing, ZIKV viremia). Examples of additionalactive ingredients that can be used to treat a mammal having, or at riskof developing, ZIKV viremia (e.g., a ZIKV infection) that can be used totreat a ZIKV infection include, without limitation, anti-histamines(e.g., chlorphenamine), corticosteroids (e.g., hydrocortisone), feverreducers (e.g., acetaminophen), immunosuppressants (e.g., mycophenolicacid), and anti-parasitics (e.g., ivermectin).

A composition including one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be designed for any route of administration.For example, a composition including one or more non-nucleoside RNApolymerase inhibitors can be designed for parenteral (e.g.,intraperitoneal) administration. Compositions suitable for parenteraladministration include, without limitation, aqueous and non-aqueoussterile injection solutions that can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient. For example, a composition includingone or more non-nucleoside RNA polymerase inhibitors can be designed fororal administration. Compositions suitable for oral administrationinclude, without limitation, liquids, tablets, capsules, pills, powders,gels, and granules. In some cases, a composition including one or morenon-nucleoside RNA polymerase inhibitors can be formulated for oraladministration.

A composition including one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be designed for any type of release (e.g.,release of the one or more non-nucleoside RNA polymerase inhibitors fromthe composition) into the mammal the composition is administered to(e.g., a mammal having, or at risk of developing, ZIKV viremia). Forexample, a composition including one or more non-nucleoside RNApolymerase inhibitors can be designed for immediate release, slowrelease, or extended release.

A composition including one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be administered to a mammal (e.g., a human)in need thereof (e.g., a mammal having, or at risk of developing, ZIKVviremia) in any appropriate dose(s). Effective doses can vary dependingon the level of ZIKV viremia, the risk of developing ZIKV infection, theroute of administration, the age and general health condition of themammal, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents, and the judgment ofthe treating physician. For example, in cases where a compositionincludes TPB, the composition can include from about 5 mg TPB perkilogram (kg) body weight of the mammal being treated to about 25 mg TPBper kg body weight of the mammal being treated (e.g., from about 7 mg/kgto about 25 mg/kg, from about 10 mg/kg to about 25 mg/kg, from about 12mg/kg to about 25 mg/kg, from about 15 mg/kg to about 25 mg/kg, fromabout 18 mg/kg to about 25 mg/kg, from about 20 mg/kg to about 25 mg/kg,from about 22 mg/kg to about 25 mg/kg, from about 5 mg/kg to about 23mg/kg, from about 5 mg/kg to about 20 mg/kg, from about 5 mg/kg to about17 mg/kg, from about 5 mg/kg to about 15 mg/kg, from about 5 mg/kg toabout 12 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 5 mg/kgto about 8 mg/kg, from about 8 mg/kg to about 22 mg/kg, from about 10mg/kg to about 20 mg/kg, from about 12 mg/kg to about 17 mg/kg, fromabout 10 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 20mg/kg TPB). In some cases, a composition including TPB can include about25 mg/kg TPB. For example, in cases where a composition includes TPB,the composition can be effective to achieve from about 100 ng of TPB permilliliter (mL) plasma in the mammal being treated to about 1000 ng ofTPB per mL plasma in the mammal being treated (e.g., a plasmaconcentration of from about 200 ng/mL to about 1000 ng/mL, from about250 ng/mL to about 1000 ng/mL, from about 275 ng/mL to about 1000 ng/mL,from about 300 ng/mL to about 1000 ng/mL, from about 350 ng/mL to about1000 ng/mL, from about 400 ng/mL to about 1000 ng/mL, from about 450ng/mL to about 1000 ng/mL, from about 500 ng/mL to about 1000 ng/mL,from about 550 ng/mL to about 1000 ng/mL, from about 600 ng/mL to about1000 ng/mL, from about 650 ng/mL to about 1000 ng/mL, from about 700ng/mL to about 1000 ng/mL, from about 750 ng/mL to about 1000 ng/mL,from about 800 ng/mL to about 1000 ng/mL, from about 850 ng/mL to about1000 ng/mL, from about 900 ng/mL to about 1000 ng/mL, from about 100ng/mL to about 900 ng/mL, from about 100 ng/mL to about 800 ng/mL, fromabout 100 ng/mL to about 700 ng/mL, from about 100 ng/mL to about 600ng/mL, from about 100 ng/mL to about 500 ng/mL, from about 100 ng/mL toabout 400 ng/mL, from about 100 ng/mL to about 300 ng/mL, from about 200ng/mL to about 900 ng/mL, from about 300 ng/mL to about 800 ng/mL, fromabout 400 ng/mL to about 700 ng/mL, from about 500 ng/mL to about 600ng/mL, from about 200 ng/mL to about 400 ng/mL, from about 400 ng/mL toabout 600 ng/mL, or from about 600 ng/mL to about 800 ng/mL TPB). Insome cases, a composition including TPB can achieve a plasmaconcentration of greater than 500 ng/mL TPB (e.g., a plasmaconcentration of about 550 ng/mL, about 600 ng/mL, about 650 ng/mL,about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL,about 900 ng/mL, or about 950 ng/mL TPB). An effective amount of acomposition including one or more non-nucleoside RNA polymeraseinhibitors can be any amount that reduces the severity and/or reduces oreliminates one or more symptom of a ZIKV infection without producingsignificant toxicity to the mammal. The effective amount can remainconstant or can be adjusted as a sliding scale or variable dosedepending on the mammal's response to treatment. Various factors caninfluence the actual effective amount used for a particular application.For example, the frequency of administration, duration of treatment, useof multiple treatment agents, route of administration, level of ZIKVviremia, severity of the ZIKV infection, and risk of developing a ZIKVinfection may require an increase or decrease in the actual effectiveamount administered.

A composition including one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be administered to a mammal (e.g., a human)in need thereof (e.g., a mammal having, or at risk of developing, ZIKVviremia) in any appropriate frequency. The frequency of administrationcan be any frequency that reduces the severity of the ZIKV infectionand/or reduces or eliminates one or more symptoms of the ZIKV infectionwithout producing significant toxicity to the mammal. For example, thefrequency of administration can be from about once a day to about tentimes a day, from about three times a day to about eight times a day, orfrom about four times a day to about six times a day. The frequency ofadministration can remain constant or can be variable during theduration of treatment. As with the effective amount, various factors caninfluence the actual frequency of administration used for a particularapplication. For example, the effective amount, duration of treatment,use of multiple treatment agents, route of administration, level of ZIKVviremia, severity of the ZIKV infection, and risk of developing a ZIKVinfection may require an increase or decrease in administrationfrequency.

A composition including one or more non-nucleoside RNA polymeraseinhibitors (e.g., TPB) can be administered to a mammal (e.g., a human)in need thereof (e.g., a mammal having, or at risk of developing, ZIKVviremia) for any appropriate duration. An effective duration foradministering a composition including one or more biguanides can be anyduration that reduces the severity of the ZIKV infection and/or reducesor eliminates one or more symptoms of the ZIKV infection withoutproducing significant toxicity to the mammal. For example, the effectiveduration can vary from several days to several months or years to alifetime. In some cases, the effective duration for the treatment ofmammal in need thereof can range in duration from about 2 days to abouta week. Multiple factors can influence the actual effective durationused for a particular treatment. For example, an effective duration canvary with the frequency of administration, effective amount, use ofmultiple treatment agents, route of administration, level of ZIKVviremia, severity of the ZIKV infection, and risk of developing a ZIKVinfection.

In some cases, methods described herein also can include administeringto a mammal in need thereof (e.g., a mammal having, or at risk ofdeveloping, ZIKV viremia) one or more additional treatments used totreat a mammal having, or at risk or developing, ZIKV viremia (e.g., aZIKV infection). The one or more additional treatments used to treat aZIKV infection can include any appropriate treatment. In some cases, aZIKV infection treatment can include getting plenty of rest. In somecases, a ZIKV infection treatment can include drinking fluids (e.g., toprevent dehydration). In some cases, a ZIKV infection treatment caninclude not taking aspirin and/or other non-steroidal anti-inflammatorydrugs (NSAIDS). In some cases, a ZIKV infection treatment can includeadministration of one or more pharmacotherapies such as antibiotics(e.g., metronidazole and dexamethasone), anti-histamines (e.g.,chlorphenamine), corticosteroids (e.g., hydrocortisone), and/or feverreducers (e.g., acetaminophen). For example, a mammal having, or at riskof developing, ZIKV viremia (e.g., a ZIKV infection) can be administeredone or more non-nucleoside RNA polymerase inhibitors (e.g., TPB) and canbe administered one or more additional treatments used to treat a ZIKVinfection. In cases where a mammal having, or at risk of developing,ZIKV viremia (e.g., a ZIKV infection) is treated with one or morenon-nucleoside RNA polymerase inhibitors and is treated with one or moreadditional agents used to treat a ZIKV infection, the additionaltreatment used to treat a ZIKV infection can be administered at the sametime or independently. For example, when administered independently, theone or more non-nucleoside RNA polymerase inhibitors can be administeredfirst, and the one or more additional treatment used to treat a ZIKVinfection can be administered second, or vice versa.

In certain instances, a course of treatment and the severity of one ormore symptoms related to the condition being treated (e.g., a ZIKVinfection) can be monitored. Any appropriate method can be used todetermine whether or not the severity of one or more symptoms is reducedor eliminated. For example, the severity of a ZIKV infection can beassessed using any appropriate methods and/or techniques, and can beassessed at different time points. For example, physical examinationscan be used to determine the severity of one or more symptoms of a ZIKVinfection.

In some cases, one or more non-nucleoside RNA polymerase inhibitors(e.g., TPB) can be used to treat a mammal having a disease or disorderassociated with a ZIKV infection. Examples of diseases and disordersassociated with a ZIKV infection include, without limitation,Guillain-Barre syndrome.

In some cases, one or more non-nucleoside RNA polymerase inhibitors(e.g., TPB) can be used to treat a mammal having, or at risk ofdeveloping, one or more additional infections caused by a member of theFlaviviridae family, which includes Dengue viruses, West Nile viruses,yellow fever viruses, and Japanese encephalitis viruses.

This document also provides kits that can be used for a variety ofapplications including, without limitation, diagnosing a mammal ashaving, or as being at risk of developing, ZIKV viremia (e.g., a ZIKVinfection); treating a mammal having, or at risk of developing, ZIKVviremia (e.g., a ZIKV infection); and/or preparing a composition (e.g.,by combining reagents) for use in diagnosing and/or treating a mammalhaving, or at risk of developing, ZIKV viremia (e.g., a ZIKV infection).In some cases, a kit provided herein can include one or morenon-nucleoside RNA inhibitors (e.g., TPB) as described herein. Forexample, a kit can include a composition (e.g., a pharmaceuticallyacceptable composition) including one or more non-nucleoside RNAinhibitors. For example, a kit can include one or more non-nucleosideRNA inhibitors and one or more pharmaceutically acceptable carriers(e.g., additives, fillers, vehicles, and/or diluents) for preparingand/or administering a composition (e.g., a vaccine composition). Insome cases, a kit provided herein can include reagents that can be usedto detect ZIKV infections. For example a kit provided herein can bedesigned as a diagnostic kit. For example, a kit provided herein can bedesigned as a kit to monitor treatment of a mammal having, or at risk ofdeveloping, ZIKV viremia (e.g., a ZIKV infection). For example, a kitprovided herein can be designed to include reagents that can be used todetect the presence of a ZIKV genome, or a portion thereof, and/or thepresence of one or more ZIKV polypeptides in samples (e.g., fluidsamples such as blood and urine) obtained from a mammal. In some cases,a kit provided herein also can include packaging. In some cases, a kitprovided herein also can include, instructions for use. For example,instructions for use can be provided as a separate component within thekit and/or printed directly on any packaging (e.g., packaging for thekit or packaging for a component within the kit).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: Discovery of a Non-Nucleoside RNA PolymeraseInhibitor for Blocking Zika Virus Replication Through in SilicoScreening Materials and Methods Compounds

Ten lead compounds (Table 1) were purchased from Hit2Lead Company(ChemBridge Corporation, San Diego, Calif.). Each compound was dissolvedin dimethyl sulfoxide (DMSO) to prepare stock solutions of 10 mM and 1mM and was stored at −20° C. The compound 1 (c1) used in this researchis3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB). Based on 1H NMR and LC-MS (ELSD, DAD 200-400 nm, MSD APCIpositive) analyses by the provider, the compound is ≥95% pure.Mycophenolic acid (MPA) and Ivermectin (IVM) were purchased from Sigma(St. Louis, Mo.) and resuspended in DMSO to prepare stock solutions. Allthe compounds used have ≥95% purity.

TABLE 1 Docking scores of the top 10 compounds. Compound MolecularWeight Score 1 541.108 −118.794 2 455.528 −118.36 3 525.572 −118.258 4516.638 −118.097 5 476.480 −117.319 6 539.618 −116.087 7 484.618−115.823 8 471.592 −115.789 9 471.574 −115.724 10 462.443 −114.333

Cells and Viruses

Vero (Cercopithecus aethiops, CCL-81), HTR-8/SVneo human trophoblast(CRL-3271), and NTERA-2 human embryonal carcinoma (CRL-1973) cells wereobtained from ATCC. The cells were grown and maintained in Dulbecco'smodified Eagle's medium (DMEM) containing 10% heat-inactivated fetalbovine serum (FBS) and penicillin/streptomycin (PS) in humidifiedchamber with 5% CO2 at 37° C. Zika virus strain PRVABC59 and MR766 wereobtained from Barbara Johnson and Brandy Russell at the Centers forDisease Control and Prevention, Fort Collins, Colo., USA. The viruseswere passaged once in Vero cells to prepare stocks and were stored at−80° C. in small aliquots. Titers of the stock viruses were determinedby plaque assay using Vero cells as described elsewhere (see, e.g.,Annamalai et al., 2017 J. Virol. 91:e01348-17).

Molecular Modeling and in Silico Screening

The ZIKV RdRp structure was modeled based on sequence homology usingModeller 9 program (Webb and Sali, 2014). The DENV-3 RdRp structure(PDB: 2J7U) was used as the template. In silico screening was performedusing Molegro Virtual Docker (MVD) (Molegro ApS, Aarhus, Denmark). Thedocking site was defined using a ray-tracing algorithm. This resulted ina cavity with a volume of approximately 1034 cubic Å. A receptor gridwas built within this cavity with a resolution of 0.2 Å and a radius of13 Å from the geometric center of the cavity in the ZIKV RdRp model. A100,000 compound library from ChemBridge (Chembridge DIVERSet™ ChemicalLibrary, ChemBridge Corporation, San Diego, Calif.) was used for thisvirtual screening. All structural analysis were conducted in theDiscovery Studio 4.0 (Biovia, San Diego, Calif.).

Inhibition Assays

Vero cells were seeded in a 96-well plate with the density of 6000 cellsper well. In the initial screening study, the compound (1 μM)-virus (0.1PFU/cell) mixture in virus growth medium (VGM) [DMEM containing 2% FBS,PS, 20 mM hydroxyethyl piperazine ethane sulfonic acid (HEPES), 1 mMsodium pyruvate, and non-essential amino acids] was added to the cellsand incubated for 72 h. In a separate experiment conducted using 12-wellplates, the cells were first infected with the virus at 0.1 PFU/cell andfollowing adsorption, the cells were washed twice in PBS and incubatedin VGM containing 1 μM concentrations of the drugs. The cell culturemedia were collected at 72-96 h post-infection and assayed forinfectious virus by plaque assay and viral genome copies by quantitativeRT-PCR (RT-qPCR). In all subsequent studies, cells in 12-well plateswere infected with ZIKV at MOI of 0.1 PFU/cell and following virusadsorption for 1 h at 37° C., VGM containing various concentrations TPBwas added to the cells and incubated as above. Clarified supernatantsfrom the infected cells were then used to determine infectious virus orgenome copies as above.

ATP-Based Cell Viability Assay

A modified ATP based cytopathic effect (CPE) assay was used for thisstudy based on the CPE method for anti-DENV drug development describedelsewhere (see, e.g., Che et al., 2009 Int. J. Clin. Exp. Med.2:363-373). Vero cells (approximately 30,000 per well) were seeded in ablack 96-well plate for 24 hours before the experiment. Cell monolayerswere treated with various concentrations of the drugs for 4 days at 37°C. The ATP concentration was measured following manufacturer'srecommendations using CellTiter-Glo kit from Promega (Madison, Wis.).Luminescence was recorded using a Veritas Microplate Luminometer at 420nm. The 50% cytotoxic concentration (CC₅₀) was calculated by anon-linear regression analysis of the dose-response curves.

Quantitative Real Time RT-PCR

ZIKV viral RNA was detected using RT-qPCR on a C100 Thermal Cycler andthe CFX96 Real-Time system (Bio-Rad). Viral RNA (vRNA) was extractedfrom culture supernatant using a QIAamp Viral RNA Mini kit (Qiagen) andTaqMan Fast Virus 1-Step Master Mix (Life technologies). ZIKV primersand probe (ZIKF: CCGCTGCCCAACACAAG (SEQ ID NO:1);ZIK-R:CCACTAACGTTCTTTTGCAGACAT (SEQ ID NO:2); PCR Probe: ZIK-P:AGCCTACCTTGACAAGCAATCAGACACTCAA (SEQ ID NO:3)) were used forquantitative RT-PCR (RT-qPCR) with the following parameters: 50° C. 30min, 95° C. 5 min, (95° C. 30 S, 58° C. 1 min)×40 cycles. RNA standardconcentrations were determined based on the back calculation with ODvalues and molecular weights and were generated through serial dilutionwith R²>0.95.

Pharmacokinetic (PK) Study Design

For PK studies, groups of Balb/C mice (n=3) were injectedintraperitoneally with doses of 5 mg/kg or 25 mg/kg of body weight ofTPB in 5% dextrose, plasma was collected from the animals at varioustimes post-injection and stored at −80° C. until analysis by LC-MS/MSfor TPB concentrations. Plasma drug levels were subjected tononcompartmental analysis (WinNonlin ver. 6.4 Certera Inc., Princeton,N.J.). The predicted steady-state levels >500 ng/ml were estimated usinga twelve h dosing of 25 mg/kg dose of the compound in mice.

Determination of Drug Concentration in Plasma

TPB was dissolved in DMSO at 1 mg/ml. Working standard solutions werethen prepared in 50% methanol in water from the stock solution.Standards (an eight-point calibration curve) and quality controls (atthree levels) were prepared by spiking the working standard solutions toblank mouse plasma. One hundred μl aliquot of plasma was mixed with 25μl of internal standard spiking solution (rilpivirine 1000 ng/ml in 50%acetonitrile in water), 1.5 ml ethyl acetate was added and vortexedvigorously for 15 min. The tubes were centrifuged at 1700×g for 5 minand 1.3 ml supernatant was evaporated to dryness under a stream ofnitrogen at 40° C. The dried extract was reconstituted with 0.1 ml of50% acetonitrile in water and 5 μl was injected into the LCMS/MSinstrument. The dynamic range of the method was 25-4000 ng/ml.

An Agilent 1200 HPLC system (Agilent Technologies, CA, USA) coupled withAB Sciex API 3200 Q Trap with an electrospray ionization source (AppliedBiosystems, Foster City, Calif., USA) was used. The mass transitions m/z541.2→330.2 and 541.2→212.2 for analyte and m/z 367.2→195.2 for internalstandard were monitored. Chromatographic separation was carried out onPhenomenex Synergi Polar-RP (150×2.0 mm, 4μ) column with isocraticmobile phase consisting of 0.1% formic acid in water (A) and 0.1% formicacid in acetonitrile (B) (20:80 v/v) at a flow rate of 0.5 ml/min. Theretention times of analyte and internal standard were 2 and 1.2 minrespectively.

Viral Inhibition Test in Mice

Balb/C mice were obtained from the Jackson Laboratory (Bar Harbor, Me.,USA). After acclimatization for four days, groups of animals (n=6 pergroup) were injected intraperitoneally with 25 mg/kg body weight dose ofTPB diluted in saline or saline alone (no drug control). Following threeinjections at 12 h intervals, 500 PFU of PRVABC59 virus diluted in PBSwas inoculated into each mouse by the subcutaneous (SC) route. Blood wascollected by retro-orbital bleeding under anesthesia at days 2, 3, 4, 5,and 6 post-infection. Viral genome copies in the plasma were determinedby RT-qPCR.

Statistical Analysis

Data were analyzed using GraphPad Prism software version 6.0. Unpairedtwo-tailed Student's t-test for pairwise comparison between the groupsto determine significant differences in viral loads (RNA levels andinfectious titer) was performed. Data were represented as means (±SEM).

Results In Silico Screening of a Compound Library Against ZIKVPolymerase (RdRp) Loops

Since the crystal structure of ZIKV RdRp was not available when thisproject was initiated, we generated a three-dimensional model of ZIKVRdRp through structural modeling based on the analog of DENV-3 RdRpstructure (PDB: 2J7U). The choice of the DENV-3 RdRP structure as thetemplate was due to its high level of protein sequence homology (65%identity and 78% similarity) and the high resolution (1.8 Å) of thestructure. The predicted ZIKV RdRp structure superimposed closely with arecently solved crystal structure (see, e.g., Duan et al., 2017 EMBO J.36:919-933) of ZIKV RdRp (FIG. 8) with C-alpha atom RMSD of 2.519. Thetarget site appears to fit well and the relative larger RMSD valueshould be mainly from the flexible loops and the outer layers of thethree domains. Like RdRp structures in other flaviviruses, the ZIKV RdRpstructure model showed a very similar right-handed architecture withfingers, palm and thumb subdomains (FIG. 1A). Subsequently, we conductedin silico screening of a library of 100,000 small molecule compoundsagainst the catalytic active site on the ZIKV RdRp molecule. The activesite is in the palm subdomain which is critical for de novo RNAsynthesis performed by ZIKV RdRp. Based on the in silico screening data,the top 10 compounds with highest docking scores are shown in Table 1.The molecular weights of these compounds are also similar (around 500Da) which are in the appropriate range of druggable compounds.

Cell-Based Inhibition Test of the Lead Compounds Against ZIKV Infection

Examination of PRVABC59 ZIKV growth in Vero cells in the presence of 1μM concentrations of the compounds (c1-c10) showed that c1,3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB, FIG. 1B), exhibited the highest inhibitory activity among the 10lead compounds tested. While the ZIKV growth was inhibited (asdetermined by genome copies in the culture supernatants) by >99% incells treated with TPB compared to the vehicle-only treated cells (FIG.2A), c6 and c10 also inhibited virus growth by nearly 70-80%. Infectiousvirus yield was inhibited by at least 1000-fold in the presence of 1 μMc1 (FIG. 2B) whereas c6 and c10 inhibited the yield by nearly 10-fold atthe same concentrations. Although the majority of the compounds could bereadily seen bound to the target site, c1, c6, and c10 appeared to havemade additional contacts with the priming loop as well as other regionsin the RdRp target site (FIG. 9). From molecular docking analysis, itappears that c1 interacts with residues in the target site of the viralRdRp (FIG. 1C-D). Three hydrogen bonds of TPB are in direct contact withtwo aspartic acid residues (D535 in motif A and D665 in motif C) in RdRp(FIG. 1C). Since these two aspartic acid residues as well as D665 arehighly conserved residues in the target and active site of all RdRps offlaviviruses and play critical roles in coordinating divalent metal ions(Mg++), TPB could potentially be a highly promising anti-ZIKV as well asanti-flavivirus drug candidate. So, from the initial cursory screeningstudies, TPB was shown to inhibit ZIKV replication significantly.

We then tested the inhibitory activity of TPB in a dose-dependent mannerin the μM range. The results show that even at 0.5 μM concentration ofTPB, significant inhibitory activity against ZIKV replication wasobserved. Both genome copy numbers (FIG. 3A) and infectious virus (FIG.3B) in the supernatants were reduced by over 100-fold at thisconcentration of TPB. Although TPB at 1 μM reduced virus growth by over1000-fold, further increase in TPB concentration did not result infurther inhibition (FIG. 3A-B). Viral E protein synthesis in infectedcells was also significantly inhibited at 0.5 μM TPB and wasundetectable at higher concentrations (FIG. 3C). These results suggestthat TPB is a potent inhibitor of ZIKV replication.

ZIKV Growth Inhibition of by TPB

Since we used the contemporary isolate of ZIKV (PRVABC59, isolated froma patient in Puerto Rico in 2015) in our initial studies, we wanted todetermine if TBP also has antiviral activity against the historicalisolate of the virus. Our results suggest that the MR766 Ugandan isolatewas also sensitive to inhibition by TPB at the concentrations tested(FIG. 4A). The extent of MR766 virus growth inhibition appeared to besimilar to that of the PRVABC59 virus (FIGS. 3 and 4A). Overall, itappears that maximal ZIKV growth inhibition by TPB could be achieved at1 or 2 μM concentrations and further increase had no significantinhibitory effect, indicating that the TPB inhibitory target issaturable at these concentrations. In addition, not only TPB inhibitedZIKV growth in Vero cells (FIG. 3), but also it inhibited the virusgrowth in other cells such as human trophoblast cell line HTR-8 (FIG.4B) as well as the human testicular cell line NTERRA (FIG. 4C) that areknown to be the targets of ZIKV infection in humans. Overall, thesestudies suggest that TBP inhibits both the contemporary and historicalisolates of the virus and that the inhibition is not cell-typedependent.

Characterization of TPB Antiviral Activity In Vitro: IC50 and CC50

Inhibitory Concentration 50 (IC50) Determination.

To characterize the anti-ZIKV potency of TPB, we conducted studies todetermine the inhibitory concentration 50 (IC50). We used serial 2-folddilutions of TPB and treated the Vero cells infected with PRVABC59. Theculture supernatants were assayed for infectious virus by plaque assayand expressed as % virus yield relative to the virus yield without TPB.The data were statistically analyzed and the IC50 concentration wasdetermined to be about 94 nM (FIG. 5A). The IC50 value of TPB in the10-100 nM range also suggests that TPB is a strong inhibitor of ZIKV anda potential drug candidate for further development.

Cellular Cytotoxicity 50 (CC50) Determination.

Low level of cellular cytotoxicity is an essential criterion for drugdevelopment. It also suggests whether the drug's inhibitory effect isindependent of cellular cytotoxicity due to the presence of the drug.Therefore, we conducted cell viability assay to determine the cellularcytotoxicity 50 (CC50) concentration of TPB. Our results show that CC50of TPB is 19.4 μM (FIG. 5B). The selectivity index 50 (SI50, CC50/IC50)is calculated to be 206. This high SI50 also suggests that TBP is notonly a potent inhibitor of ZIKV at sub-micromolar concentrations but isalso nontoxic to the cells.

Comparison of TPB Inhibition with Other Known Inhibitors of ZIKV.

To further compare the potency of TPB relative to other identified ZIKVinhibitors, two inhibitors were examined that have been recently shownto inhibit ZIKV replication. Mycophenolic acid (MPA) is animmunosuppressant drug used to prevent rejection in organtransplantation and was shown to inhibit DENV RNA replication (see,e.g., Diamond et al., 2002 Virology 304:211-221). In a screen ofFDA-approved drugs for inhibition of ZIKV infection, MPA at 1 μM wasfound to inhibit infection of cells in vitro by ZIKV by over 99% (see,e.g., Barrows et al., 2016 Cell Host Microbe 20:259-270). Likewise,Ivermectin (IVM), an anti-parasitic drug was found to inhibit ZIKVinfection strongly at 10 μM (see, e.g., Barrows et al., 2016 Cell HostMicrobe 20:259-270). A side-by-side comparison of the inhibitory potencyof TPB with MPA and IVM shows that while TPB inhibited ZIKV yield byover 1000-fold, MPA and IVM inhibited virus yield by approximately 10-to 20-fold (FIG. 6). These results suggest that TPB is more potent ininhibiting ZIKV as compared to MPA or IVM.

Antiviral Activity of TPB In Vivo Since TPB was found to be a potentinhibitor of ZIKV replication in vitro, we wanted to examine if it alsoinhibits virus replication and viremia in an immunocompetent mousemodel. Therefore, a pharmacokinetics (PK) analysis of TPB inimmunocompetent Balb/C mice was conducted to examine the stability andin vivo retention of the drug. The results of PK studies suggest thatTPB is retained in the mouse plasma at approximately 100 ng/ml level10-12 h post-injection at the two doses tested (FIG. 7A). Based onnon-compartment analysis of the data, it was estimated that steady-statelevels >500 ng/ml of TPB (˜1 μM) could be achieved using a twelve hourdosing at 25 mg/kg dose of the compound in mice. To examine the effectof the drug on ZIKV growth in mice, groups of mice (n=6) were injectedwith the drug at 25 mg/kg dose and subsequently infected with 500 PFU ofZIKV. Virus load in the plasma of the animals at 24 hour intervals wasdetermined. Results of virus growth (genome copies) in individualanimals (FIG. 7B) show that these immunocompetent mice supportedtransient ZIKV growth and the level of viral RNA detected on day 4post-infection was nearly 40-fold lower in mice injected with the drugas compared to the group injected with the vehicle (5% dextrose) alone(FIG. 7C). The level of TPB in the plasma on average reached nearly 270ng/ml by 2 days post-infection (FIG. 7D). Although this level of TPB wasnot optimal for maximal virus growth inhibition as observed under invitro conditions, the results suggest that TPB exerts significant growthinhibition of ZIKV in vivo.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a mammal having a Zikavirus (ZIKV) infection, said method comprising: administering to saidmammal a composition comprising a non-nucleoside RNA polymeraseinhibitor.
 2. The method of claim 1, wherein said mammal is a human. 3.The method of claim 1, wherein said non-nucleoside RNA polymeraseinhibitor can bind to a catalytic active site of an RNA-dependentRNA-polymerase (RdRp) of said ZIKV to inhibit ZIKV replication.
 4. Themethod of claim 1, wherein said non-nucleoside RNA polymerase inhibitoris3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB).
 5. The method of claim 1, wherein said non-nucleoside RNApolymerase inhibitor has an inhibitory concentration 50 (IC₅₀) of fromabout 10 nM to about 200 nM.
 6. The method of claim 1, wherein saidnon-nucleoside RNA polymerase inhibitor has a cytotoxicity concentration50 (CC₅₀) of from about 15 μM to about 25 μM.
 7. The method of claim 1,wherein said non-nucleoside RNA polymerase inhibitor has a selectiveindex 50 (SI₅₀) of about
 206. 8. The method of claim 1, wherein saidadministering step is performed prior to said ZIKV infection or aftersaid ZIKV infection.
 9. The method of claim 1, wherein saidadministering step is performed prior to said ZIKV infection and afterinfection said ZIKV infection.
 10. The method of claim 1, wherein saidnon-nucleoside RNA polymerase inhibitor is administeredintraperitoneally, intravenously, intramuscularly, or subcutaneously.11. A method of preventing microcephaly in a fetus said methodcomprising: administering a composition comprising a non-nucleoside RNApolymerase inhibitor to a mammal pregnant with said fetus, wherein saidmammal has a ZIKV infection.
 12. The method of claim 11, wherein saidmammal is a human.
 13. The method of claim 11, wherein saidnon-nucleoside RNA polymerase inhibitor is3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB).
 14. A method of treating an adult mammal having Guillain-Barresyndrome said method comprising: administering to said mammal acomposition comprising a non-nucleoside RNA polymerase inhibitor,wherein said mammal has a ZIKV infection.
 15. The method of claim 14,wherein said mammal is a human.
 16. The method of claim 14, wherein saidnon-nucleoside RNA polymerase inhibitor is3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB).
 17. A composition for reducing Zika virus (ZIKV) viremia within amammal, said composition comprising a non-nucleoside RNA polymeraseinhibitor.
 18. The composition of claim 17, wherein said non-nucleosideRNA polymerase inhibitor is3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide(TPB).
 19. The composition of claim 17, wherein said non-nucleoside RNApolymerase inhibitor can bind to a catalytic active site of an anRNA-dependent RNA-polymerase (RdRp) of said ZIKV.
 20. The method ofclaim 17, wherein said non-nucleoside RNA polymerase inhibitor has aninhibitory concentration 50 (IC₅₀) of from about 10 nM to about 200 nM.21. The method of claim 17, wherein said non-nucleoside RNA polymeraseinhibitor has a cytotoxicity concentration 50 (CC₅₀) of from about 15 μMto about 25 μM.
 22. The method of claim 17, wherein said non-nucleosideRNA polymerase inhibitor has a selective index 50 (SI₅₀) of about 206.23. The composition of claim 17, further comprising a pharmaceuticallyacceptable carrier.